DETECTION OF HIV BY A MAGNETIC BEAD BASED VIRAL CAPTURE ASSAY

一种从体液中检测艾滋病病毒的新技术正在开发中 如血清、血浆和感染细胞的培养上清液。H9 用HIV-MN株感染细胞， 在第4天收集。 在上清液中测量p24抗原。 病毒 存在于流体中的颗粒通过用 抗体包被的珠子。 均匀尺寸的颗粒主要被包覆 与抗IgG抗体反应，再与抗p24和抗gp 120抗体反应 两者都是IgG类抗体。孵育16小时后 在低温下放置10小时后，用缓冲液洗涤珠粒并在4 ℃下储存。 然后将培养物上清液与珠粒一起在低温下孵育24小时。 16小时将珠洗涤两次并用胍处理 异硫氰酸盐缓冲液以剥离病毒。来自剥离的RNA 制备材料并用于使用SK 38/ 39引物的RT-PCR， 扩增产物与P-32标记的SK 19探针杂交， 液相杂交技术在我们的初步实验中， 用涂有抗HIV抗体的磁珠富集， 在放射自显影印迹上显示强阳性信号。 该方法 适用于检测感染者血清和血浆中的病毒 个人，以获得敏感性和特异性。 这 技术将用于检测病毒以及表征 通过PCR测序直接从珠粒中分离病毒。

A novel technique is being developed for detecting HIV from body fluids such as serum, plasma and culture supernatants of infected cells. H9 cells were infected with HIV-MN strain and the culture supernatants collected on day 4. p24 antigen was measured in the supernatant. Virus particles present in the fluids were concentrated by capture with antibody coated beads. Particles of uniform size were coated primarily with anti IgG antibodies and then reacted with anti p24 and anti gp120 separately, both being IgG class antibodies. After incubation for 16 hours in the cold, the beads were washed with buffer and stored at 4 C. Culture supernatants were then incubated with the beads in the cold for 16 hours. The beads were washed twice and treated with guanidine isothiocyanate buffer to strip off the virus. RNA from the stripped material was prepared and used for RT-PCR using SK 38/ 39 primers and amplified products were hybridized with P-32 labelled SK 19 probe by liquid hybridization technique. In our preliminary experiments, virions were enriched with magnetic beads coated with anti HIV antibodies and showed strong positive signals on an autoradiographic blot. This method is being adapted to detecting virus in the serum and plasma of infected individuals in an effort to derive sensitivity and specificity. This technique will be of use in detecting virus as well as in characterizing virus isolates by PCR sequencing directly from the bead.